Effect Of Extracellular Vesicle Deliveried-circSCMH1 On Functional Recovery Of Ischemic Stroke And The Underlying Machanisms

Aims:Stroke is a leading cause of adult disability that can severely compromise patients’quality of life in the worldwide,yet no effective medication currently exists to accelerate rehabilitation.A variety of circular RNA（circ RNA）molecules are known to function in ischemic brain injury,and screening of circ RNA with diagnostic and therapeutic prospects is of great significance.However,how to safely and effectively deliver the circ RNA to the targeted site remains to be resolved.Thus,the study sought to search the related circ RNA in the pathogenesis of stroke,and then exlpore the effective therapeutic strategies for brain repair after ischemia stroke based on extracellular vesicles（EVs）.Methods:circ RNA was screened from the plasma of acute ischemic stroke（AIS）patients using circ RNA microarray and valided in AIS and photothrombotic（PT）mice combined with quantitative polymerase chain reaction（q PCR）.Clinical statistic was used to analyze the relationship between circ SCMH1 and the severity of AIS,the potential in accessing the diagnosis and prognosis of AIS.Engineered rabies virus glycoprotein（RVG）-circ SCMH1-EVs based on modified RVG-lamp2b-EVs targeting brain were generated to deliver circ SCMH1.Nanoparticle tracking analysis（NTA）,transmission electron microscope（TEM）,western blot and q PCR were used to characterize RVG-circ SCMH1-EVs.HE staining and flow cytometry were used to examine the toxicity and immunogenicity,respectively.Behavioral tasks were performed to evaluate the effect of RVG-circ SCMH1-EVs on sensorimotor functions in rodent models.Nissl staining and magnetic resonance imaging（MRI）were used to examine the effect of RVG-circ SCMH1-EVs on infarct size.Golgi staining combined with Sholl analysis,lentivirus transduction,immunostaining,and flow cytometry were used to explore the cellular mechanism（s）of brain recovery.Proteomic assay and RNA-seq combined with transcriptional profiling and molecular profiling were used to identify the molecular mechanism（s）of brain recovery.Results:circ RNA SCMH1（circ SCMH1）levels were significantly decreased in plasma of AIS patients and PT mice,as well as in the peri-infarct zone of mice.Statistical analysis revealed that the baseline circ SCMH1 levels on the 1^st day were low related to AIS with a significant area under the curve（AUC）of 0.7896 in diagnosing AIS.Furthermore,there was a higher level of the baseline circ SCMH1 on the 1^st day between patients that eventually achieved good outcomes versus those with poor outcomes,and the level rose along with stroke recovery.ROC curve analysis was performed to calculate the predictive power of baseline circ SCMH1 levels for stroke outcome and the AUC was 0.6864.The engineered EVs were physically homogenous,with a size distribution peaking at 117 nm in diameter.Brain targeting signals were expressed on the surface of EVs,and circ SCMH1 was overexpressed in the EVs.circ SCMH1 was safely and effectively delivered to the peri-infarct zone via the engineered RVG-circ SCMH1-EVs.The study identified that RVG-circ SCMH1-EVs improved functional recovery of PT mice with no significant difference in infarct volume between RVG-circ SCMH1-EV group and RVG-Vector-EV group.However,the protective effect could not be ruled out as the infarct volume was decreased in distal middle cerebral artery occlusion（d MCAO）and transient middle cerebral artery occlusion（t MCAO）model mice.Furthermore,the study discovered that RVG-circ SCMH1-EVs relieved the damage to spines,branches,denderic lengenth and intersections,enhanced neuronal plasticity;and also inhibited astrocytes and microglia activation in the peri-infact area,as well as peripheral immune cell infiltration in PT mice,including CD4⁺T cells、CD8⁺T cells、B220⁺B cells and Ly6G^-Ly6C^lo cells.Elucidating a molecular mechanism that circ SCMH1bound to the transcription factor methyl-Cp G binding protein 2（Me CP2）,releasing repression of Me CP2 target gene transcription,thereby promoting brain repair after stroke.Conclusions:Based on data obtained from the plasma of AIS patients,using the RVG-Lamp2b-EV delivery system,this study reveals that RVG-circ SCMH1-EVs afford multiple advantages to promote functional recovery in the rodent stroke models,and demonstrates the basic mechanism of how circ SCMH1 can be therapeutically exploited to improve post-stroke outcomes.This study will provide the basis for the development of treatment that effectively promote the recovery of ischemic patients.